Increasing the stability of recombinant adult human apohemoglobin

Abstract

The disclosure relates to recombinant adult human apohemoglobin (apo-rHb) in which the stability has been increased by replacement of an amino acid with a counterpart from another organism, such as a deep sea diving mammal. This mutated apo-rHb may be more stable and/or give higher production yields than unmutated adult human apo-rHb. The mutated apo-rHb may be produced in microorganisms, such as E. coli or yeast cells, or animal erythroid cells. Some apo-rHb of the present disclosure may be used as part of a blood substitute.

1. A method of producing a stabilized apohemoglobin subunit comprising modifying at least two to up to 15 amino acids of the amino acid sequence of adult human apohemoglobin subunit to match a counterpart from an apohemoglobin subunit from a deep sea diving mammal, wherein a stabilized apohemoglobin subunit exhibiting improved resistance to degradation or precipitation prior to heme insertion as compared to unmodified adult human apohemoglobin is produced. 
     
     
       2. A method according to  claim 1 , wherein said deep sea diving mammal is a sperm whale. 
     
     
       3. A method according to  claim 1 , wherein said modifying comprises forming a nucleic acid encoding an adult human apohemoglobin subunit with at least two to up to 15 variant amino acids that match their counterpart amino-acids of an apohemoglobin subunit from a deep sea diving mammal and expressing said nucleic acid in  E. coli , another microorganism, or animal erythroid cells. 
     
     
       4. A recombinant adult human apohemoglobin production cell comprising:
 a nucleic acid encoding an adult human apohemoglobin subunit with at least two to up to 15 variant amino acids that match their counterpart amino acids of an apohemoglobin subunit from a deep sea diving mammal that is more resistant to degradation or precipitation prior to heme insertion than an unmodified adult human apohemoglobin subunit; and 
 other cellular components sufficient to produce a recombinant adult human apohemoglobin. 
 
     
     
       5. A production cell according to  claim 4 , wherein the deep sea diving mammal is a sperm whale. 
     
     
       6. A production cell according to  claim 4 , wherein the production cell is an  E. coli  cell, a cell from another microorganism, or an animal erythroid cell. 
     
     
       7. A production cell according to  claim 4 , further comprising an expressible nucleic acid comprising the nucleic acid encoding an adult human apohemoglobin subunit. 
     
     
       8. A system for recombinant adult human apohemoglobin production comprising:
 a plurality of production cells; and 
 a nucleic acid encoding a stabilized recombinant adult human apohemoglobin subunit more resistant to degradation or precipitation prior to heme insertion than unmodified adult human apohemoglobin; 
 wherein the nucleic acid encodes an adult human apohemoglobin subunit with at least two to up to 15 variant amino acids that match their counterpart amino acids of an apohemoglobin subunit from a deep sea diving mammal. 
 
     
     
       9. A system according to  claim 8 , further comprising a second nucleic acid encoding a second recombinant adult human apohemoglobin subunit. 
     
     
       10. A system according to  claim 8 , further comprising a second nucleic acid encoding a wild-type adult human apohemoglobin subunit. 
     
     
       11. A system according to  claim 8 , wherein the system produces recombinant adult human apohemoglobin that may be used as part of a blood substitute product.